The present invention relates to an enzyme detection device and, more specifically, an enzyme detection device for detecting an enzyme capable of modifying a provided substrate that is recognisable by a specific binding molecule in either its modified or unmodified state. The invention also relates to a method of detecting an enzyme capable of modifying a substrate.
There are many clinical and research situations where it is useful to be able to detect the presence, in a sample, of an enzyme that has a particular catalytic activity. One example of such an enzyme is a protease, which digests proteins by hydrolysing and cleaving peptide bonds. Thus a protein which is degraded by a protease is cleaved into two or more smaller peptides. Another example is a sialidase which will cleave a sialic acid moiety from a larger carbohydrate.
A diagnostic test kit is disclosed in US2006/0003394 which detects an enzyme in a sample. The kit comprises “reactive complexes” which each comprise a target ligand joined to a reporter and a specific binding member. The target ligand is cleavable by the enzyme to be detected in order to release the reporter. The mixture of the reactive complexes and the sample is subsequently applied to a chromatographic medium. The chromatographic medium comprises a first detection zone which is capable of capturing the specific binding member on the target ligand and a second detection zone which his capable of binding the reporter. Therefore, in use, the mixture of the sample and reactive complexes is applied to the chromatographic medium and flows firstly through the first detection zone and then through the second detection zone. At the first detection zone the target ligand is captured. Any enzyme in the sample cleaves the reporter from the target ligand and the cleaved reporter is captured at the second detection zone, leaving any un-cleaved reporter remaining attached to the target ligand, at the first detection zone. Therefore, the presence of the enzyme in the sample is determined by the presence of the reporter at the second detection zone (or the absence of the reporter from the first detection zone). One problem with the kit disclosed in US2006/0003394 is that the kit can tend to give inaccurate results because the enzyme continues to be active while the mixture is flowing along the chromatographic medium. The enzyme continues to cleave the reporter from the target ligand while the reactive complexes are immobilised at the first capture zone. Thus, the signal at the first detection zone and the second detection zone can tend to vary over time, there being no clear end point to the reaction.
Another problem with the kit of US2006/0003394 is that it can only detect an enzyme that cleaves the target ligand and is not able to detect enzymes that have other effects such as adding moieties to a target ligand (e.g. phosphorylation or glycosylation).
A further problem with the kit of US2006/0003394 is that it cannot detect the activity of enzymes that have to cleave the terminus of the target ligand, such as an exopeptidase, or an exocarbohydrase, because the requirement to join a reporter to the terminus would change the chemical nature of the terminus to an extent that would prevent interaction with the enzyme's active site.